Bioluminescence assay for the highly sensitive detection of botulinum neurotoxin A activity.

This article describes a novel bioluminescence assay for detecting the proteolytic activity of Botulinum NeuroToxins (BoNT) in complex matrices. The assay is capable of detecting traces of BoNT in blood samples as well as in food drinks. The assay was responsive to BoNT/A subtypes 1 to 5, and serotype E3 in buffered solutions. It was responsive to filtered Clostridium botulinum supernatants and BoNT/A1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fM after 4 h RT incubation and in bouillon at concentrations down to 120 fM after 21 h RT incubation. In combination with an immunocapture/enrichment step it could detect BoNT/A1 in citrated plasma at concentrations down to 30 fM (1.2 mouse LD50 per mL). The simplicity of the assay, combined with a demonstrated ability to lyophilize the reagents, demonstrates its usefulness for detection of BoNT in non-specialised analytical laboratories.

Resolving power of lens-less low energy electron point source microscopy.

Simulations show the resolving power of lens-less low energy electron point source microscopy to be approximately 15 A for electron energies of between 14 and 105 eV. This resolution can be improved to 10 A by employing a composite hologram method. However, these values fall short of predictions of at least 3 A resolution for electron energies in this range because the limited divergence angle of the electron beam had not previously been taken into account. Also shown is that electron coherence from field emitting tips that terminate in a single atom will not limit the resolving power as much as the beam divergence angle. The penetration depth of electrons with energies of between 50 and 100 eV, into biological molecules, was estimated from the inelastic mean free path length to be 25 A . This places an upper limit on the size of biological molecules for which internal structural information can be obtained using low energy electrons.

Amplified expression and large-scale purification of protein L'.

The gene fragment (PPL') encoding the functional unit of peptostreptococcus protein L was isolated using PCR and expressed in E. coli. As the gene fragment lacked its own promoter, the 5' PCR primer was designed to incorporate an Nde1 restriction site (CATATG) into the gene. This enabled the gene to be cloned in frame into an Nde1 restriction site immediately downstream of a trp promoter. To prevent read through, a stop codon was introduced into the 3' primer. Expression of PPL' was up to 27% total cell protein which compares favourably to the 0.1% total soluble cell protein obtained from the original clone of peptostreptococcus. Following a heat step homogeneous PPL' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in yields of 90%.

Rapid large-scale preparation of recombinant Erwinia chrysanthemi L-asparaginase.

L-asparaginase from Erwinia provides an alternative to the enzyme from E. coli for the effective treatment of acute lymphoblastic leukaemia. A procedure was required for the large-scale partial purification of the recombinant Erwinia enzyme cloned and expressed in Erwinia. Enzyme was extracted from Erwinia at high pH and extraneous protein precipitated at low pH. S-Sepharose FF was selected as the medium of choice for the chromatography step since it was adequate for the high flow rates required (linear flow rate 315 cm h-1) and the methylsulphonate functional groups exploited the high pI of the enzyme by allowing binding of L-asparaginase at pH 4.8 while most of the other proteins passed through the column. The useful capacity of the matrix was up to 34 mg enzyme/ml matrix at a linear flow rate of 95 cm h-1 and 15.4 mg enzyme/ml matrix at a linear flow rate of 315 cm h-1. Weakly bound protein was removed by a wash at pH 6.0. The L-asparaginase was eluted by a wash at pH 6.8 (linear flow rate 95 cm h-1) and was substantially pure, only requiring polishing steps to be suitable for use as a parenteral agent. The purity of the protein was complemented by a 92% recovery of active enzyme from this cation-exchange matrix.

Amplified expression and large-scale purification of protein G'.

PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity.

Measurement of the discomfort component of illness.

Discomfort is a prominent component of illness, but it is difficult to measure on a scale that allows its formal inclusion in a health status index. The authors tested the content validity of defining various discomforts in terms of their quality, duration, and intensity and found no responses that could not be categorized within this conceptual framework. They then analyzed the ability of patients to ascribe preference values to a sample of discomfort statements, based on this characterization of discomfort, and found them able to do this reliably using magnitude estimation. These results show that, although the universe of discomforts cannot be measured directly on a common scale, they can be compared using a scale of social preference. This will allow the formal incorporation of the discomfort component of illness into health status indexes based upon dysfunctions, discomforts, and prognosis.

Large-scale purification of the chromosomal beta-lactamase from Enterobacter cloacae P99.

Homogeneous beta-lactamase (beta-lactam hydrolase, E.C. 3.5.2.6) from Enterobacter cloacae P99, an enzyme that has an important function in antibiotic resistance, was prepared using a single cation-exchange chromatographic step with CM-Sepharose fast-flow. A 6-g amount of the enzyme was isolated from 5 kg of cell paste, with 84% of the enzyme activity in the cell homogenate being recovered by the single cation-exchange step. The specific activity of the beta-lactamase was 587 U/mg protein. The relative molecular mass of the enzyme was determined to be 45 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the isoelectric point was 8.95.

Expectation and burnout in the developmental disabilities field.

Psychological burnout was assessed in staff members at workshops and community residences for the developmentally disabled. Participants also rated expectations for client progress and for their own contribution to clients. They reported on change in expectation since they entered the field. High expectations were related to low burnout; workers who reported experiencing large negative expectation change were most burned out. Burnout seemed to be prevented when staff members made an expectation shift from reliance on client progress to a sense of personal efficacy. This finding is discussed in terms of personal causation, internal control of reinforcement, and adaptation-level theory. There was little evidence of client depersonalization, a usual component of burnout. Such a burnout pattern may be a function of the ethic of community care for the developmentally disabled.

Paired determinations of blood carboxyhemoglobin concentration and carbon monoxide excretion rate in term and preterm infants.

Paired determinations of COHb and VeCO were performed on 30 term infants (38 to 42 weeks' gestation) and 26 preterm infants (28 to 37 weeks' gestation) during the first week of life. All subjects were breathing room air at the time of the study. Values of COHb were corrected for RAco by linear regression of COHb (percent saturation) vs RAco (ppm). Regression coefficients for term and preterm infants with no history of pulmonary impairment were nearly identical (COHb = 0.175 RAco + 0.45, r = 0.77, n = 25 for term infants; COHb = 0.168 RAco + 0.51, r = 0.82, n = 9 for preterm infants) and agreed well with theoretical values. For the group of term infants, linear regression of Veco (microliter/kg/hr) vs. COHbc, where COHbc = COHb - 0.17 RAco, resulted in VEco = 23.4 COHbc + 4.02, r = 0.75, n = 30. The corresponding relationship for preterm infants with no history of pulmonary impairment was VEco = 24.7 COHbc + 3.85, r = 0.61, n = 13. For a subpopulation of preterm infants with a history of pulmonary dysfunction, the correlation decreased significantly, with VEco = 4.34 COHbc + 17.6, r = 0.097, n = 11. These results demonstrate that (1) COHbc is a reasonable index of VEco and consequently of the heme catabolic rate in both term and preterm infants with no clinical history of pulmonary dysfunction and (2) inference of VEco from COHbc may be misleading in certain cases without a consideration of the factors relating these two variables.

Pulmonary excretion rates of carbon monoxide using a modified technique: differences between premature and full-term infants.

The pathophysiology of the exaggerated hyperbilirubinemia in premature infants remains unclear. The relative contribution of bilirubin production may be estimated by measuring the pulmonary excretion rate of carbon monoxide (VeCO). We found that the mean VeCO of premature infants, 16.7 +/- 5.0 microliters/kg/h, was significantly elevated (p less than 0.05) compared with the mean VeCO of full-term infants, 13.9 +/- 3.5 microliters/kg/h. Premature infants who required phototherapy had a significantly (p less than 0.05) higher mean VeCO than those who did not. The VeCO did not correlate with gestational age, implying that factors which associate frequently but variably with gestational age may have an important influence on heme catabolism.